Louis, MO) and purified upon introduction using a previously described protocol (Sekura, 1981) to a purity greater than 99% while determined by high-pressure liquid chromatography (Sheng and Duffel, 2001)
Louis, MO) and purified upon introduction using a previously described protocol (Sekura, 1981) to a purity greater than 99% while determined by high-pressure liquid chromatography (Sheng and Duffel, 2001). protocol (Sekura, 1981) to a purity greater than 99% as determined by high-pressure liquid chromatography (Sheng and Duffel, 2001). 2-Mercaptothanol, estradiol, estradiol-sulfate, potassium phosphate, (BL21 (DE3) cells (50 for 1 hour at 4C, and the supernatant was discarded. The cell pellet was resuspended in 10 ml ice-cold bacterial lysis buffer A [10 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 for 1 hour. The cell extract (440 mg protein) was applied to a DE-52 anion exchange column (2.5 20 cm) equilibrated with buffer B [50 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose, 1 mM DTT, 10% (v/v) glycerol, and 0.05% (v/v) Tween 20] and then washed with approximately 1 L buffer B to remove COL4A5 proteins that did not bind to this column. Once the absorbance of the eluate at 280 nm experienced reached a baseline value, the hSULT1E1 was eluted having a linear gradient created between 200 ml buffer B and 200 ml buffer B comprising 0.1 M KCl. The fractions comprising hSULT1E1 were then combined and Revaprazan Hydrochloride concentrated by ultrafiltration (Amicon stirred cell having a YM10 membrane; Millipore, Bedford, MA). The concentration of potassium chloride was then reduced through successive dilution and concentration by ultrafiltration, with the dilutions carried out using the same buffer to be employed for the subsequent hydroxyapatite chromatography step [i.e., buffer C: 10 mM potassium phosphate, pH 6.8, 0.25 M sucrose, 1 mM DTT, and 0.05% (v/v) Tween 20]. The producing protein (26 mg) was applied to a column of hydroxyapatite (2.5 3.0 cm) that had been equilibrated with buffer C. Buffer C was used to wash the column and remove all nonbinding proteins, and the elution of hSULT1E1 was carried out having a linear gradient created between 80 ml buffer C and 80 ml buffer C comprising 0.4 M potassium phosphate. The fractions comprising hSULT1E1 activity were pooled and concentrated by ultrafiltration using an Amicon membrane. Approximately 10 mg Revaprazan Hydrochloride purified hSULT1E1 was recovered from your hydroxyapatite column. The subunit molecular mass of hSULT1E1 was found to be approximately 35 kDa by SDS-PAGE, which is consistent with previously reported data for this enzyme (Aksoy et al., 1994). The purity of hSULT1E1 was greater than 94% when analyzed by densitometry on SDS-PAGE. At each purification step, hSULT1E1-comprising fractions were recognized and quantitated having a previously explained methylene blue assay (Duffel et al., 1989) using 25 BL21 (DE3) cells (50 Squirewell, Duffel. Squirewell. Squirewell, Duffel. Squirewell, Duffel. Footnotes This work was Revaprazan Hydrochloride supported from the National Institutes of Health National Tumor Institute [Give R01 CA038683]. dx.doi.org/10.1124/dmd.115.063206. This short article has supplemental material available at dmd.aspetjournals.org..